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Abstract:

Lactobacillus strains that have a genetic Profile I based on Apa I, Not
I, and Xba I digests are provided. Preferably, the strains decrease level
of at least one of coliforms and E. coli within the gastrointestinal
tract of an animal. A direct-fed microbial that includes the strain is
additionally provided. A method of feeding an animal the strain and a
method of forming a direct fed microbial that includes the strain is also
provided.

Claims:

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. A method of forming a direct fed microbial, the method comprising:
(a) growing, in a liquid nutrient broth, a culture including at least one
Lactobacillus strain that has a Profile I based on Apa I, Not I and Xba I
digests, as shown in FIG. 1 and Table 6; and (b) separating the strain
from the liquid nutrient broth.

34. The method of claim 32, wherein the gel further comprises an
artificial coloring agent.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.
No. 10/624,443, filed Jul. 22, 2003, which claims priority to U.S.
Provisional Patent Application No. 60/397,654, filed Jul. 22, 2002, the
entireties of both of which are incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The invention relates to Lactobacillus strains for ingesting by
animals. More particularly, though not exclusively, the present invention
relates to Lactobacillus strains that are useful as direct-fed microbials
for pigs.

BACKGROUND OF THE INVENTION

[0003] Strains of the genus Lactobacillus are normal inhabitants of the
gastrointestinal tract of many animal species. In pigs, lactobacilli are
one of the principal bacterial groups in the proximal region of the
digestive tract (Barrow, P. A., R. Fuller, and N.J. Newport. 1977. Inft,
Immun. 18: 586-595). Their beneficial role in the intestinal tract has
been attributed to their ability to survive the digestive process, attach
to the epithelial lining of the intestinal tract, produce lactic acid and
other antimicrobial compounds, and prevent the colonization of pathogens
via competitive exclusion (Savage, D.C. 1987. Factors affecting the
biocontrol of bacterial pathogens in the intestine. Food Technol. 41:
82-87).

[0004] Many allogenic and autogenic factors influence the microbial
population of the gastrointestinal tract (Savage, D.C. 1989. Rev. sci.
tech Off. in Epiz. 8: 259-273). Allogenic factors such as alterations in
the diet and environment along with maturation of the host are major
influences on the succession of Lactobacillus strains in the
gastrointestinal tract of pigs during the post-weaning phase. Although it
has been well documented that these changes may have severe effects on
the host, little is understood about the distribution and diversity of
lactobacilli species during this period.

[0005] Current industry practices to improve health and, more
specifically, reduce the levels of coliforms and E. coli within the
gastrointestinal tract of pigs generally include feeding antibiotics at
subtherapeutic levels. However, the practice of feeding antibiotics to
livestock has raised concerns about increasing the antibiotic resistance
of microbial pathogens in the food supply.

[0006] Another approach to improving the health of animals is to alter the
inhabitants of their gastrointestinal tract. Altering the inhabitants of
the gastrointestinal tract of animals has been attempted by feeding
direct-fed microbials to animals. The efficacy of single or multiple
strains of Lactobacillus commonly used in commercial direct-fed
microbials has been and continues to be debated. This debate is primarily
due to inconsistent performance of previous direct-fed microbials. This
inconsistency may be due to the fact that many commercial direct-fed
microbials are composed of Lactobacillus strains commonly used as silage
inoculants or cheese starter cultures. These strains may be effective to
inoculate silage or to convert milk into cheese, but have no proven
efficacy as direct fed microbials for animal feeding. While the "one
strain for all products" approach may be an economical method for the
commercial fermentation industry, this does not provide the best strains
for each application.

[0007] In view of the foregoing, it would be desirable to provide a
direct-fed microbial that reduces the levels of coliforms and E. coli
within the gastrointestinal tract of pigs. In particular, it would be
desirable to provide a direct-fed microbial that provides a healthier
intestinal microflora during the weaning transition period in pigs.

SUMMARY OF THE INVENTION

[0008] The invention, which is defined by the claims set out at the end of
this disclosure, is intended to solve at least some of the problems noted
above. Lactobacillus strains that have a Profile I based on Apa I, Not I,
and Xba I digests, as shown in FIG. 1 and Table 6, are provided.
Preferably, the strains decrease levels of at least one of coliforms and
E. coli within the gastrointestinal tract of an animal. Preferred strains
include, but are not limited to L. brevis strains, L. fermentum strains,
and L. murinus strains. Useful strains of the invention have been
isolated from the pars oesophagea of a pig. A particularly preferred
strain is L. brevis strain 1E-1, although any Lactobacillus strain having
a Profile I based on Apa I, Not I, and Xba I digests, as shown in FIG. 1
and Table 6 are expected to work in the invention.

[0009] A method of feeding an animal is also provided. The method
comprises feeding the animal a Lactobacillus strain that has a Profile I
based on Apa I, Not I, and Xba I digests, as shown in FIG. 1 and Table 6.
Preferably, the strain decreases levels of at least one of coliforms and
E. coli within the gastrointestinal tract of an animal.

[0010] A direct-fed microbial is additionally provided. The direct-fed
microbial includes at least one Lactobacillus strain that has a Profile I
based on Apa I, Not I, and Xba I digests, as shown in FIG. 1 and Table 6.
The direct-fed microbial additionally includes a carrier.

[0011] Also provided is a method of forming a direct fed microbial. In the
method, a culture is grown in a liquid nutrient broth. The culture
includes at least one Lactobacillus strain that has a Profile I based on
Apa I, Not I, and Xba I digests, as shown in FIG. 1 and Table 6. The
strain is separated from the liquid nutrient broth.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Preferred exemplary embodiments of the invention are illustrated in
the accompanying drawings.

[0013] FIG. 1 shows Apa I, Not I, and Xba I digests of various strains
from pig 1, including strain 1E-1.

[0024] FIG. 12 is a graph showing the number of sulfuric goblet cells in
the duodenum of pigs on d 10, 21, and 28 of age (interaction, P<0.06).
Means within each day post-weaning with different letter designations
differ significantly (P<0.06).

[0025] Before explaining embodiments of the invention in detail, it is to
be understood that the invention is not limited in its application to the
details of construction and the arrangement of the components set forth
in the following description or illustrated in the drawings. The
invention is capable of other embodiments or being practiced or carried
out in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description and
should not be regarded as limiting.

DETAILED DESCRIPTION

[0026] In accordance with the present invention, there may be employed
conventional molecular biology and microbiology within the skill of the
art. Such techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual,
Third Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.

[0027] Described herein are Lactobacillus strains that have positive
effects on the health of animals. Preferred Lactobacillus strains will
now be described that are useful in pigs. This example is not intended to
limit the invention to Lactobacillus strains usable only in pigs. The
Lactobacillus strains of the invention are isolated from an animal, such
as a pig. Lactobacillus strains of the invention preferably reduce the
levels of coliforms and E. coli within the gastrointestinal tract of
pigs. The Lactobacillus strains also provide a healthier intestinal
microflora during a pre-weaning period in pigs. Thus, the Lactobacillus
strains provide a healthier intestinal microflora during a pre-weaning
and weaning period in pigs.

[0028] Lactobacillus strains of the invention have a profile I based on
Apa I, Not I and Xba I digests, as shown in FIG. 1 and Table 6 (below).
Preferred Lactobacillus strains include, but are not limited to, L.
brevis, L. fermentum, and L. murinus. A preferred Lactobacillus brevis
strain is 1E-1, which was isolated from the intestinal tract of a
healthy, weaned pig. Strain 1E-1 is available from the microorganism
collection of the American Type Culture Collection, 10801 University
Blvd., Manassas, Va. 20110, under accession number PTA-6509, and was
deposited on Jan. 12, 2005.

[0029] The Lactobacillus strains of the invention can be used as a
direct-fed microbial. In a preferred embodiment, the direct-fed microbial
is L. brevis strain 1E-1. Furthermore, the multiple Lactobacillus strains
can be combined as a direct-fed microbial.

[0030] Characterization and Screening of Lactobacillus Strains:

[0031] In one exemplary evaluation of the bacteria of the present
invention, the intestinal tracts of thirteen weaned pigs, ten healthy and
three with scours, were sampled for the Lactobacillus strains found
therein. As is detailed in Example 1 below, twenty-five numerically
dominant isolates were selected from samples of the pars oesophagea,
duodenum, jejunum, and ileum from each pig.

[0032] Isolates were identified using biochemical and carbohydrate
fermentation tests. Plasmid profiling and pulsed-field gel
electrophoresis (PFGE) were used to attempt to distinguish between
strains of lactobacilli within a species. Higher numbers of lactobacilli
were detected throughout the intestinal tract of healthy pigs when
compared to the intestinal tract of sick pigs. The highest lactobacilli
counts for both healthy and sick pigs were found in the pars oesophagea
samples (healthy pigs-1.3×108 CFU/g and sick
pigs-1.6×106 CFU/g). The lowest counts were found in the
jejunal samples for both groups (healthy pigs-1.9×106 CFU/g
and sick pigs-4.3×105 CFU/g).

[0033] Biochemical identification of the isolates indicated that the
lactobacilli populations of healthy pigs were much more homogeneous than
lactobacilli populations of sick pigs. In healthy pigs, the majority of
the isolates were identified as L. brevis. Depending on the location, L.
brevis accounted for 35-90% of the lactobacilli population. Similar, but
not always, identical plasmid profiles were observed among L. brevis
isolates. Identical plasmid profiles were observed among isolates
identified as different species. PFGE was useful in identifying
individual strains within a species.

[0034] Preparation and Feeding of Lactobacillus Strains:

[0035] A direct-fed microbial of the invention includes a Lactobacillus
strain that has a Profile I based on Apa I, Not I and Xba I digests, as
shown in FIG. 1 and Table 6. A preferred strain is the L. brevis strain
1E-1, although other Lactobacillus strains having a Profile I can be
used. A carrier can be added to the direct-fed microbial. The carrier can
be a liquid carrier, a solid carrier, or any other suitable carrier. A
preferred liquid carrier is a milk replacer. Milk replacers are typically
milk substitutes in powdered form that are mixed with water to form a
composition that resembles milk. Another preferred liquid carrier is
water. Dry carriers include, but are not limited to, animal feed.

[0036] The Lactobacillus strains of the present invention may be presented
in various physical forms, for example, as a top dress, as a water
soluble concentrate for use as a liquid drench or to be added to a milk
replacer or gels. In a preferred embodiment of the top dress form of the
Lactobacillus strains, a freeze-dried Lactobacillus strain fermentation
product is added to a carrier, such as whey, limestone (calcium
carbonate), rice hulls, yeast culture, dried starch, or sodium silico
aluminate.

[0037] In a preferred embodiment of the water soluble concentrate for a
liquid drench or milk replacer supplement, a freeze-dried Lactobacillus
strain fermentation product is added to a water soluble carrier, such as
whey, maltodextrin, sucrose, dextrose, dried starch, or sodium silico
aluminate, and a liquid is added to form the drench or the supplement is
added to milk or a milk replacer. In a preferred embodiment of the gels
form, a Lactobacillus strain fermentation product is added to a carrier,
such as one or more of vegetable oil, sucrose, silicon dioxide,
polysorbate 80, propylene glycol, butylated hydroxyanisole, citric acid,
and ethoxyquin to form the gel. An artificial coloring can be added to
the gel.

[0038] Particularly preferred ways of feeding the direct-fed microbial
include a milk supplement (replacer) fed during lactation 7-19 days prior
to weaning, a single dose of a gel paste or drench given 1-2 days prior
to weaning followed by dosing in water systems in the nursery for 7 days,
and a single does of a gel paste or drench given 1-2 days prior to
weaning followed by dosing in gruel feed in the nursery for 2-3 days. The
direct-fed microbial can be fed in other forms, for differing periods of
time, and at different stages in the pig's weaning.

[0039] Typically, the direct-fed microbial is formed by growing a culture
including the Lactobacillus strain of choice in a liquid nutrient broth.
The Lactobacillus strain of the culture is then separated from the liquid
nutrient broth, such as by centrifugation. The Lactobacillus strain can
then be freeze-dried. The freeze-dried Lactobacillus strain can be added
to a carrier. This addition can be done immediately or at a subsequent
time. Where the freeze-dried Lactobacillus strains are added at a
subsequent time, they are preferably stored in a waterproof container,
such as a foil pack.

[0040] The Lactobacillus strains of the invention can be fed to an animal.
In a preferred embodiment, the animal is fed a Lactobacillus brevis
strain 1-E1. Particularly useful results have been obtained when young
pigs, including pre-weaning pigs, weaned pigs, and post-weaned pigs, are
fed one or more the Lactobacillus strain of the invention.

[0041] Preferably, the animal is fed the Lactobacillus strain such that
the amount of Lactobacillus strain delivered to the animal is about
1×108 CFU to about 1×1010 CFU per day. More
preferably, the animal is fed the Lactobacillus strain such that the
amount of Lactobacillus strain delivered to the animal is about
5×109 CFU per day. However, it should be noted that higher and
lower doses of the Lactobacillus strain can be fed to the animal and are
believed to have a positive effect on the animal.

[0042] As is shown below in detail, feeding the Lactobacillus strain of
the invention to animals altered the intestinal flora of the animals,
decreasing levels of coliforms and E. coli in the animals. Maintaining a
normal healthy intestinal microflora during the profound environmental
and nutritional changes at weaning is critical to ensure optimal
performance for pigs. Feeding the Lactobacillus strain of the invention
also increased the average daily gain, increased the villus:crypt ratio
in the animals, and decreased the number of sulfuric goblet cells, as is
also shown in detail below.

[0044] The effects of feeding L. brevis strain 1E-1 on the
gastrointestinal microflora of pre-weaning, weaning, and post-weaning
pigs have been determined. As is detailed below in Example 2, sows and
gilts were randomly assigned to one of three treatments. In Example 2,
four litters received no milk replacer (control), five litters received
milk replacer, and five litters received milk replacer supplemented with
strain 1E-1.

[0045] In Example 2, coliforms and E. coli were enumerated from pars
oesophageal, duodenal, jejunal, and ileal regions of intestinal tracts
from one pig per litter at 9-13 days of age (pre-weaning) and at 19-23
days of age (weaning). In pre-weaning pigs, E. coli and coliform
populations in pars oesophageal, duodenal, and ileal regions of
pre-weaning pigs were not significantly different in the three groups. As
is shown in more detail in Example 2, pigs receiving strain 1E-1 had
significantly lower jejunal E. coli populations compared to control
(P<0.02) and milk replacer (P<0.05). Jejunal coliform populations
tended to be lower for pigs receiving strain 1E-1 compared to control
pigs (P<0.12) but were not significantly different compared to pigs
receiving milk replacer. There were no treatment effects on populations
of coliforms and E. coli in the pars oesophageal and duodenal regions for
pigs at weaning. Pigs receiving strain 1E-1 had significantly lower E.
coli populations in the jejunal region compared to control (P<0.01)
and milk replacer (P<0.11). There were no significant treatment
effects on jejunal coliform populations for pigs at weaning. In the ileal
region of weaning pigs, the coliform populations neared significance for
pigs receiving strain 1E-1 when compared to control (P<0.07). E. coli
populations were significantly lower for pigs receiving strain 1E-1
compared to control pigs (P<0.05) and pigs receiving milk replacer
(P<0.02). These results show that feeding strain 1E-1 provides a
healthier intestinal microflora during lactation.

[0046] The use of L. brevis strain 1E-1 is shown herein to reduce the
levels of coliforms and E. coli within the gastrointestinal tract of
pigs, providing a healthier intestinal microflora during the pre-weaning
period. The strongest response shown by strain 1E-1 was in the distal
regions of the gastrointestinal tract more than the proximal regions,
within pigs at weaning (19-23 days old) more than the pre-weaning pigs
(9-13 days old), and against E. coli more than coliforms.

[0047] In addition, feeding the Lactobacillus strain to animals improves
the health of the animal. For instance, feeding the Lactobacillus strain
to young pigs decreases the incidence of scours in the young pigs.

[0048] Additionally, as is detailed below in Example 3, populations of E.
coli and coliforms in the small intestine were reduced pre- and
post-weaning when pigs were supplemented with 1E-1.

[0049] Intestinal morphology improved when animals were fed the
Lactobacillus strain of the invention. For instance, villus:crypt ratio
was greater and the number of sulfuric goblet cells was less with
supplementation with the Lactobacillus strain. These data indicate that
supplementing with strain 1E-1 and other strains having a Profile I based
on Apa I, Not I and Xba I digests, as shown in FIG. 1 and Table 6
pre-weaning improves nursery performance and provides a healthier
intestinal environment. An increase in weight at weaning in pigs fed the
Lactobacillus strain was also observed.

EXAMPLES

[0050] The following Examples are provided for illustrative purposes only.
The Examples are included herein solely to aid in a more complete
understanding of the presently described invention. The Examples do not
limit the scope of the invention described or claimed herein in any
fashion.

Example 1

Characterization of the Predominant Lactobacilli Isolated from the
Intestinal Tract of Post-Weaned Pigs

Materials and Methods

[0051] Pigs:

[0052] Thirteen crossbred pigs raised in a commercial facility in Arkansas
were used in this study. After weaning at 21 days, pigs were fed a
complex Phase 1 prestarter diet. At 7-10 days post-weaning, 3-5 pigs were
selected from either a healthy group or a group of pigs identified as
having scours and transported to Oklahoma State University. Pigs were
killed by exsanguation and samples of the pars oesophagea, duodenum,
jejunum, and ileum were aseptically removed along with 25 g samples of
fecal and stomach contents. Three repetitions of this procedure were
completed for a total sample size of 13 pigs (10 healthy and 3 with
scours).

[0056] Isolates picked from LBS plates were confirmed as lactobacilli by
the Gram-stain reaction, cell morphology, and catalase reaction. The
species identity of isolates was determined using API Rapid CH kits
(Analytab Products, Plainview, N.Y.) according to the manufacturer's
directions. Fermentation patterns were observed and recorded for each
isolate at 24, 36, and 48 h. Fermentation patterns of each isolate were
compared to differential characteristics provided in Bergey's Manual for
species identification.

[0057] Plasmid DNA Isolation:

[0058] Plasmid DNA was isolated from the lactobacilli strains as follows:
a 1% inoculum taken from a 24 hour culture was placed into 10 ml of
sterile MRS broth and incubated at 37° C. until the optical
density (660 nm) reached 0.8 (log phase). Cell suspensions were then
harvested by centrifugation (12,000×g for 15 min). The supernatant
was decanted and the pellet resuspended in 1 ml of Tris-EDTA buffer
containing 15% sucrose. Resuspended cells were stored in 1.5 ml
centrifuge tubes at -20° C. until plasmid DNA analysis was
performed. Frozen samples were allowed to thaw at room temperature. Cells
were washed by harvesting (12,000×g for 5 minutes) and resuspending
the pellet in 1 ml of Tris-EDTA buffer containing 15% sucrose. After
washing, the pellet was resuspended to a final volume of 250 μl with
fresh Tris-EDTA-sucrose buffer and mixed well by vortexing. Lysozyme (50
μl of a 60 mg/ml solution) was added, and the tubes were incubated on
ice for 1 hour. Pronase (10 mg/ml: pre-incubated at 37° C. for 1
hour) was added (35 μl) followed by incubation at 37° C. for 30
minutes.

[0059] Following incubation, 0.25 M EDTA was added (111 μl) to the
sample and the tubes held for 15 minutes on ice. Tris-EDTA containing 20%
SDS was added (111 μl) and held on ice for an additional 15 minutes.
Sodium acetate (75 μl of a 3.0 M solution) was added followed by a 30
minute incubation on ice. Debris was pelleted by centrifugation
(12,000×g for 15 minutes) and the supernatant transferred to a
clean 1.5 ml microcentrifuge tube. Cold ethanol (750 μl of 95%) was
added to the tube containing the supernatant and mixed well by gently
inverting the tube several times. The samples were stored at -20°
C. for 1 hour to precipitate the DNA. DNA was pelleted by centrifugation
(12,000×g for 15 minutes) and allowed to dry. The DNA was
resuspended in 40 μl Tris-EDTA buffer, 5 μl of tracking dye was
added and the mixture loaded onto an agarose gel. DNA was separated by
gel electrophoresis using a 0.7% agarose gel at 50 volts. Agarose gels
were examined after a 45 minute staining period in ethidium bromide
solution.

[0060] Preparation of Intact Genomic DNA:

[0061] Intact genomic DNA from representative strains was isolated from
cells embedded in agarose beads using a modification of the method of
Rehberger, T. G. 1993. Curr. Microbiol. 27: 21-25. Cultures were grown to
mid-log stage in MRS broth, harvested by centrifugation (9,000×g
for 10 min), and resuspended to one-tenth the original volume in ET
buffer (50 mM EDTA, 1 mM Tris-HCl, pH 8.0). The cell suspension was mixed
with an equal volume of 1% low-melting point agarose (Beekman
Instruments, Palo Alto, Calif.), loaded into a syringe and injected into
tygon tubing where it was allowed to solidify. The solidified
cell-agarose mixture was forced into cold ET buffer and gently vortexed
to break the string into smaller bead like pieces. The beads were
resuspended in 10 ml of 10×ET buffer containing 5 mg/ml of lysozyme
and incubated on ice for 2 hours to digest the cell wall material.

[0062] After incubation, the beads were harvested by centrifugation
(4,000×g for 10 min) and resuspended in 10 ml of lysis buffer
(10×ET buffer containing 100 ug/ml of proteinase K and 1%
Sarkosyl), followed by incubation at 55 C for 5-7 hours to lyse the cells
and release the genomic DNA. After cellular lysis, the beads were
harvested by centrifugation (4,000×g for 10 min), resuspended in 10
ml of 1 mM phenylmethylsulfonyl fluoride, and incubated at room
temperature for 2 hours to remove contaminating protease activity. The
beads containing the purified DNA were washed three times in TE buffer
(10 mM Tris-HCl, 1 mM EDTA-Na2, pH 7.5), resuspended in 10 ml of TE
buffer and stored at 4° C. until restriction endonuclease
digestion.

[0064] Agarose beads containing DNA were equilibrated in 1×
restriction endonuclease buffer for 1 hour before enzyme digestion. After
the beads were equilibrated, 10-20 units of the restriction enzyme were
added to 90 ul of beads and incubated at the appropriate temperature for
6-8 hours. Following digestion, the enzymes were inactivated by heating
for 10 minutes at 65° C. This melted the beads and allowed for
easy loading onto the gel for fragment separation.

[0065] DNA fragments were separated on 1.0% agarose gels in 0.5×TBE
buffer at 15° C. for 20 hours using a CHEF-DRIII electrophoresis
system (Bio-Rad, Hercules, Calif.). Each set of restriction endonuclease
digests were separated at different initial and final pulse times to
provide maximum separation of small, medium, and large fragments. To
determine the molecular size of the DNA fragments lambda DNA multimers,
intact yeast chromosomes and restriction fragments of lambda DNA were
included as standards.

Results and Discussion

[0066] Higher mean numbers of lactobacilli populations were detected in
all gastrointestinal samples from healthy pigs compared to pigs with
scours (Table 1). However, the variation in the lactobacilli populations
among healthy pigs for all sample locations was greater than the
difference seen between healthy and sick pigs.

[0067] Independent of the health of the animal, differences in
lactobacilli populations were observed among different regions in the
gastrointestinal tract. The par oesophageal region of all animals
contained the highest number of lactobacilli compared to other
gastrointestinal regions. The jejunal region of all animals contained the
lowest number of lactobacilli compared to other gastrointestinal regions.

[0068] Identification of the predominant Lactobacillus species from the
digestive tract samples is shown in Table 2. In some cases, the
predominant species accounted for 100% of the total lactobacilli
population. L. brevis and L. murinus were found to be the most common
predominant species in healthy pigs while L. plantarum and L. murinus
were found to be most common in pigs with scours.

[0069] The predominant Lactobacillus species identified for each region of
the digestive tract (Table 3) was found to be different between healthy
and sick pigs. L. brevis was found to be the most common predominant
species in three regions of healthy pigs while L. plantarum and L.
murinus were found to be most common in two regions each of pigs with
scours.

[0070] The predominant Lactobacillus species identified from each pig
examined (Table 4) indicated that L. brevis was the predominant species
in 3 of the 5 healthy pigs and L. plantarum and L. murinus were the
predominant species in pigs with scours.

[0071] Plasmid profiling was used in an attempt to distinguish strains of
lactobacilli. Strains were assigned to a plasmid profile type for
comparison to other strains from different regions and pigs. Table 5
lists the seven major profile types observed in this study. All plasmid
profiles types were found to be common to two or more pigs and two or
more regions. However, no profile type was shared among healthy and sick
pigs. Fewer number of isolates were examined (140) for plasmids from sick
pigs, which may have affected this observation. Plasmid profile type I
was the most common profile in healthy pigs, while type III was most
common in pigs with scours. Plasmid profiling was not as discriminatory a
typing technique as genomic DNA fingerprinting to distinguish between
strains within a species.

[0072] Comparisons of genomic DNA fingerprints produced by restriction
endonuclease digestion of intact genomic DNA were used to determine the
genetic relatedness among strains (data not shown). In general, a
majority of strains isolated from the same animal were found to have
identical Apa I, Not I, and Xba I fingerprints. Populations of
lactobacilli in different gastric regions were composed of similar
strains. To date, no evidence was found indicating distinct populations
for the different regions of the gastrointestinal tract examined in this
study. In contrast, distinct populations were identified that were
specific for healthy and sick pigs. These findings indicate a distinct
difference in the dominant strains of the lactobacilli populations
between healthy and sick pigs.

[0073] Pulsed-field gel electrophoresis was useful at identifying
differences among phenotypically indistinguishable strains. As an
example, at least three different L. brevis strains and three different
L. murinus have been identified from the isolates examined from pig 1
(Table 6). In addition, genomic fingerprints have also been found to be
identical between phenotypically different strains. This may be due to
genetic changes in the genes responsible for the carbohydrate
fermentation(s) found to distinguish the strains biochemically. These
changes could have resulted in the loss of function but may not have
altered the restriction sites or the distances between them and
therefore, go undetected as differences by genomic fingerprints.

Influence of Lactobacillus brevis Strain 1E-1 on the Gastrointestinal
Microflora and Performance of Pre-Weaning and Weaning Pigs

[0074] From the study described in Example 1, it was determined that the
intestinal tract of healthy pigs had higher levels of lactobacilli.
Genetic analysis of the lactobacilli found in healthy pigs indicated a
homogenous population of strains, whereas the lactobacilli populations
found in the sick pigs were heterogeneous. The majority of the isolates
(59%) were identified as a single genotype (Profile I based on Apa I, Not
I and Xba I digests) that was biochemically identified as L. brevis. This
strain is now referred to as 1E-1 and is the Lactobacillus strain used in
this example. Lanes 1 and 14 contain a Lambda concatamer as a molecular
weight (MW) marker. FIG. 1 shows Apa I digests (left hand lane), Not I
digests (middle lane), and Xba I digests (right hand lane) of various
strains, including strain 1E-1. All Lactobacillus strains with a Profile
I based on Apa I, Not I, and Xba I digests are expected to work in the
invention.

Materials and Methods

[0075] Sows and gilts were blocked by parity and sire and randomly
allotted to one of three treatments as they were placed in the farrowing
room at 110-112 days of gestation. Litters, starting at birth, received
no supplemental milk replacer (control), supplemental milk replacer
(18.5% solids, 1.5 lbs/gallon) without 1E-1 (milk), or supplemental milk
replacer (18.5% solids, 1.5 lbs/gallon) with 1E-1 (milk plus 1E-1). Pigs
received treatments up to the day of weaning. At weaning, pigs within
each group were ranked by weight. A phase I diet, fed for the first two
weeks post-weaning, contained 3.75% spray-dries plasma and at least 15%
lactose. A phase II diet, fed until the completion of the study (28
days), contained 1.0% plasma, 1.5% blood cells, and at least 8% lactose.

[0076] The milk replacer system used in this study was an in-line system.
The milk replacer was supplied to the pigs ad libitum in a small bowl
supplied by a central 30-gallon tank. The tank was equipped with a hydro
pump and a pressure regulator that pumped the milk replacer to the pens
as needed. A baby pig nipple inside each bowl allowed milk to flow into
the bowl only when touched by a pigs nose. This was used to minimize
spillage and waste of the milk replacer. The entire system was flushed on
a daily basis with hot water to remove spoiled milk or sediment, and
fresh milk was prepared using a commercial milk replacer (Merrick's
Litter-Gro, Union Center, Wis.).

[0077] Whole intestinal tracts were removed from one randomly selected pig
per litter at 9-13 days of age and from another at 19-23 days. Tracts
were immediately placed in a Whirl-pak® bag containing approximately
200 ml sterile phosphate buffer (0.3 mM KH2PO4, 1 mM
MgSO4, 0.05% cysteine hydrochloride, pH=7.0). Tracts were sent to
Agtech Products, Inc. for further analysis, which included enumerating E.
coli and coliforms, harvesting the bacterial community in the tracts, and
community DNA isolation to trace 1E-1 throughout the tracts.

[0078] Whole tracts were aseptically cut into pars oesophageal, duodenal,
jejunal, and ileal sections, and each section was rinsed with sterile
phosphate buffer until all contents were washed out. The section was cut
lengthwise to expose the epithelial lining, and the sterile rinse was
repeated. The weight of each section was recorded and the section placed
in a new Whirl-pak® bag. Sterile phosphate buffer (99 ml) was added
to each bag and masticated for 60 seconds. Each sample was plated on VRB
(Difco Sparks, Md.) for the enumeration of coliforms and CHROMagar
(CHROMagar Paris, France) for the enumeration of E. coli. Spiral plating
techniques were used at 10-1 and 10-3 dilutions on the
Autoplate 4000 (Spiral Biotech, Inc., Norwood, Mass.). The remaining
liquid was poured into a sterile 250 ml centrifuge bottle and the
intestinal scrapings harvested by centrifugation at 8000 rpm for 15
minutes. The supernatant was carefully removed and the pellet was
resuspended with 10 ml MRS+10% glycerol. The sample was then transferred
to a sterile 15 ml Falcon® tube and stored at -20° C. until
DNA isolation.

[0079] DNA was isolated from the harvested cells using a High Pure PCR
Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany).
1E-1 was then isolated within the community DNA by using PCR on the
16S-23S intergenic spacer region of each sample. Specific primers were
used that annealed to conserved regions of the 16S and 23S genes
(Tilsala-Timisjarvi, A., and T. Alatossava. 1997 Development of
oligonucleotide primers from the 16S-23S rRNA intergenic sequences for
identifying different dairy and probiotic lactic acid Lactobacillus
strain by PCR. Int. J. Food Microbiol. 35: 49-56). PCR was performed
following the procedures of Tannock et al. (1999. Identification of
Lactobacillus isolates from the gastrointestinal tract, silage, and
yoghurt by 16S-23S rRNA gene intergenic spacer region sequence
comparisons. 65: 4264-4267). PCR mixtures contained 5 μl of 10×
polymerase buffer (Boehringer Mannheim), 200 μM each deoxynucleoside
triphosphate, 80 pM each primer, 8 μl DNA, and 2.6 U of Expand High
Fidelity PCR System (Boehringer Mannheim GmbH, Mannheim, Germany) DNA
polymerase in a total volume of 50 μl. The PCR program began with a
pre-incubation at 94° C. for 2 min., then 95° C. for 30 s,
55° C. for 30 s, and 72° C. for 30 s. This was repeated for
30 cycles and finished with a 5-min incubation at 72° C. PCR
products were then isolated by electrophoresis in a 1% agarose gel and
visualized by UV transillumination after being stained in ethidium
bromide solution.

Results

Gastrointestinal Microflora:

[0080] Pre-Weaning Pigs: There were no significant treatment effects on
populations of coliforms within the pars oesophageal, duodenal, and ileal
sections of pre-weaning pigs (FIG. 2). The jejunal coliform populations
tended to be lower for pigs receiving milk replacer plus 1E-1 compared to
control pigs (P<0.12) but were not significantly different when
compared to pigs receiving milk replacer alone.

[0081] E. coli populations enumerated within the pars oesophageal,
duodenal, and ileal sections were not significantly different among
pre-weaning pigs receiving the three treatments (FIG. 3). However, in the
ileal section, there was a trend for lower levels of E. coli in the pigs
receiving milk plus 1E-1 compared to control pigs and those receiving
milk replacer alone. Pigs receiving milk plus 1E-1 had significantly
lower jejunal E. coli populations compared to control (P<0.02) and
milk replacer alone (P<0.05).

[0082] Weaning Pigs: The coliform levels within the pars oesophageal,
duodenal, and jejunal regions of weaning pigs showed no significance
between treatments (FIG. 4). Coliform levels in the ileal region,
however, tended to be lower for pigs receiving milk replacer plus 1E-1
when compared to control (P<0.07), but were not significantly
different when compared to milk replacer alone.

[0083] The E. coli levels in the weaning pigs showed some large
differences between treatments within the distal regions of the
gastrointestinal tract (FIG. 5). There were no significant treatment
effects on E. coli levels within the pars oesophageal region. In the
duodenum, E. coli levels tended to be lower for pigs receiving milk
replacer plus 1E-1 compared to control (P<0.13). Within the jejunal
region, pigs receiving milk replacer plus 1E-1 had significantly lower E.
coli populations compared to the control (P<0.01) and the difference
in E. coli populations was nearing significance compared to milk replacer
alone (P<0.11). The E. coli populations in the ileal region showed a
significant reduction in E. coli levels for pigs receiving milk replacer
plus 1E-1 when compared to control (P<0.05) and milk replacer alone
(P<0.02).

[0084] Overall, the microbial data showed a reduction in coliforms and E.
coli for pre-weaning and weaning pigs receiving milk replacer plus 1E-1.
The most noticeable response was in the distal regions of the
gastrointestinal tract compared to the pars oesophageal and duodenal
(proximal) regions. The reduction in E. coli levels in the pigs was
greater than the reduction in the level of coliforms. A greater
difference was also observed between treatments in pigs at weaning
compared to pre-weaning pigs.

[0085] The establishment of strain 1E-1 in the gastrointestinal tract was
studied using the 16S-23S intergenic spacer region of 1E-1 (Tannock, G.
W., et al. 1999. Identification of Lactobacillus isolates from the
gastrointestinal tract, silage, and yoghurt by 16S-23S rRNA gene
intergenic spacer region sequence comparisons. Appl. Environ. Microbiol.
65: 4264-4267). The intergenic spacer region has been known to be highly
variable between lactobacilli. Tannock's results indicated that this was
a relatively simple and rapid method by which lactobacilli can be
identified without resorting to the use of species-specific primers. The
results of this analysis, however, showed identical PCR products between
native lactobacilli and strain 1E-1. Even a Cfo I restriction digest did
not distinguish between these isolates. Therefore, the use of the 16S-23S
intergenic spacer region has not been useful for tracing strain 1E-1
within the gastrointestinal tract.

Performance Data:

[0086] Suckling Pigs:

[0087] The suckling performance data is shown below in Table 7. Pigs
receiving strain 1E-1 had a significant increase in average daily gain
(ADG) from birth to weaning when compared to the control (P=0.07), but
not significantly different when compared to pigs fed milk replacer
alone. At five days to weaning and ten days to weaning, pigs receiving
strain 1E-1 showed a significant increase in ADG when compared to control
(P=0.05 and P=0.06, respectively), but not when compared to milk replacer
alone. There was no significant difference in weight at weaning for pigs
receiving any of the treatments.

[0089] The nursery pig performance data is shown below in Table 8. Nursery
pig performance was monitored to determine the long-term effects of the
treatments. In phase 1, the pigs receiving strain 1E-1 had a significant
increase in ADG when compared to the pigs receiving milk replacer alone
(P<0.05), but were not significantly different compared to pigs fed no
milk (control). In phase 2, a significant improvement in the gain:feed
ratio was observed for pigs receiving milk alone compared to control pigs
(P<0.05), however, there was no significant difference when compared
to pigs receiving strain 1E-1. Overall (phase 1 and 2 combined), there
was no significant difference for ADG or gain:feed among the treatments.
Pigs fed strain 1E-1 had a significantly higher weight at the end of
phase 1 compared to pigs receiving milk replacer alone (P=0.07), but the
weight was not significantly higher compared to control. At the end of
Phase 2, the pigs receiving milk plus strain 1E-1 tended to have a higher
weight when compared to control pigs (P=0.11), but the weight was not
different compared to the milk replacer alone.

[0090] In each experiment, litters were allotted to two treatments at
farrowing: either a control milk supplement, or the control milk
supplement containing strain 1E-1. The milk supplement contained 18.5%
solids. Treatments were administered throughout the lactation period.
After weaning, pigs were grouped 6 pigs per pen.

[0091] Coliforms and E. coli were enumerated from pars oesophageal,
duodenal, jejunal, and ileal regions of the enteric tracts, and gut
morphology was assessed from one pig/litter at approximately 10
(pre-weaning) and 22 (weaning) days of age, and after weaning at 28 days
of age. Gut morphology was examined to determine villus height and area,
crypt depth and the different mucins (neutral, acidic, and sulfuric)
produced from enteric goblet cells. Duodenum and ileum tissue samples
were taken and sectioned at 4-6 μm. Sections were mounted on
polylysine-coated slides and stained with 1) hematoxylin and eosin, 2)
alcian blue and periodic acid schiff, and 3) high iron dye. Intestinal
microflora populations were determined for pars oesophageal, duodenal,
jejunal, and ileal sections. Samples from these regions were processed
and each sample was plated. VRB was used for enumeration of coliforms.
CHROMagar was used for enumeration of E. coli. Spiral plating techniques
were used at 10-1 and 10-3 dilutions on an Autoplate 4000.

Results and Discussion

[0092] Growth Performance:

[0093] Strain 1E-1 supplementation did not affect pig growth performance
during the pre- or post-weaning periods. The lack of a performance
response with strain 1E-1 supplementation in this Example is likely a
consequence of lower coliform and E. coli populations in all regions of
the gastrointestinal tract from all ages of pigs examined in this
experiment. In Example 3, coliform and E. coli levels in pre-weaning pigs
ranged from 100 to 1000 times lower than coliform and E. coli levels in
pigs from Example 2. At weaning, coliform and E. coli levels in pigs from
Example 3 were 1000 times lower in the proximal regions of the
gastrointestinal tract and 10 to 100 times lower in the distal regions
compared to pigs in Example 2. Although the coliform and E. coli levels
were reduced by feeding L. brevis 1E-1 in Example 3, pig performance was
not significantly improved due to decreased pathogenic challenge.

[0096] Intestinal Morphology: As is shown in FIG. 10, pigs provided strain
1E-1 had greater (P<0.01) ileal villus:crypt ratio at 10 days of age
compared to control pigs, although there was no difference at 22 and 28
days of age (interaction, P<0.05). The greater ileal villus:crypt
ratio indicates that strain 1E-1 increases the maturation of the distal
region of the gut in 10 day old pigs. FIG. 11 shows that pigs provided
strain 1E-1 had greater (P<0.01) duodenum villus:crypt ratio at 22
days of age compared to control pigs, although there was no difference at
10 and 28 days of age (interaction, P<0.02).

[0097] The number of duodenal sulfuric goblet cells was less (P=0.06) when
pigs were provided strain 1E-1 compared to control pigs at 10 days of
age, although there was no difference at 22 and 28 days of age
(interaction, P=0.06; FIG. 12). Sulphomucins are normally absent from the
small intestine, but can be produced by crypt goblet cells when the small
intestinal mucosa is altered (Specian, R. D. and M. G. Oliver. 1991.
Functional biology of intestinal goblet cells. Am. J. Physiol.
260:C183-C193). The decrease in the number of duodenal sulfuric goblet
cells by strain 1E-1 in pigs at 10 days of age may be an indication of a
healthier gastrointestinal tract. In addition, the lower number of
sulfuric goblet cells, combined with the increase in villus:crypt ratio
in strain 1E-1-supplemented pigs suggests that strain 1E-1 affords some
protection from the intestinal disruption that occurs at weaning.

[0098] It is understood that the various preferred embodiments are shown
and described above to illustrate different possible features of the
invention and the varying ways in which these features may be combined.
Apart from combining the different features of the above embodiments in
varying ways, other modifications are also considered to be within the
scope of the invention. The invention is not intended to be limited to
the preferred embodiments described above, but rather is intended to be
limited only by the claims set out below. Thus, the invention encompasses
all alternative embodiments that fall literally or equivalently within
the scope of the claims.